The nuclear envelope mediates a remarkable transport process. Precursor RNAs are retained in the nucleus, while processed messenger RNA, transfer RNA and ribosomal subunits are transported to the cytoplasm. Proteins destined for the nucleus become localized soon after synthesis and again following mitosis, while cytoplasmic proteins are excluded. My previous work on the kinetics of protein import demonstrated that protein import to the nucleus is mediated by a limiting cellular component. A novel feature of this work was the demonstration that synthetic peptide nuclear localization signals, when crosslinked to carrier proteins and microinjected into the cytoplasm of Xenopus oocytes, are transported into nuclei. The aim of this proposal is to extend the kinetic analysis of nuclear transport in higher eukaryotes. We will test the hypothesis that all macromolecular nuclear trafficking processes share at least one component of a general nucleocytoplasmic trafficking apparatus. Because rate constants for only a single karyophilic protein (peptide-BSA) have been determined we will first repeat these experiments using a number of artificial and natural transport substrates in Xenous oocytes. In addition to synthetic peptide signals, we will use other more natural substrates such as genetically engineered fusion proteins containing signal sequences, and endogenous nuclear proteins. Whether or not any of these substrates share steps along the cell's trafficking pathway will be determined by classical competition analysis. Because protein import and tRNA export are both saturable and have similar rate constants, we aim to determine whether they share a common rate-limiting component. Previously we found that anti-signal sequence antibodies recognize a family of endogenous nuclear proteins which localize to nucleoli. Basic biochemical and trafficking properties of these novel proteins will be studied. We will test the hypothesis that P38, a major nuclear signal binding protein also present in nucleoli and identified by its strong binding to a signal sequence affinity column, governs the nucleolar localization of these signal- containing proteins. Finally, we propose to learn how the central channel of the nuclear pore complex is gated. We are combining a new imaging technique, which includes cryoelectron microscopy of frozen- hydrated specimens and single particle image analysis, with colloidal gold labeling techniques. Amphibian oocytes are microinjected with colloidal gold coated with karyophilic proteins and lectins known to bind nuclear pore proteins. The labeled nuclei are extruded into buffer and processed for electron microscopy. Preliminary results suggest that the central channel physically dilates many tens of angstroms to admit large gold substrates. The pattern of lectin-labeled gold binding reveals the changing configuration of channel-rim proteins in response to this dilation. These studies will be completed and extended.